The goal of the proposed research is to define the regulation of normal and mutant cardiac muscle Ca2+ release channels (ryanodine receptors, RyR2s) under normal conditions as well as those in ischemic and post-ischemic heart. The cardiac Ca2+ release channel is a 30S protein complex comprised of four 560 kDa RyR2 subunits and four 12.6 kDa FK506 binding protein (FKBP12.6) subunits. Multiple endogenous effector molecules and post-translational modifications regulate RyR2, including Ca2+, Mg2+, ATP, calmodulin, protein phosphorylation, and thiol oxidation/reduction and S-nitrosylation. The principal hypotheses to be tested in the proposed research are that RyR2 is differentially regulated by calmodulin and by redox activation pathways involving free thiol groups. These hypotheses will be addressed using biochemical and electrophysiological methods and by creating mutant embryonic stem (ES) cell lines and mice. The Specific Aims are: (1) Characterize the regulation of RyR2 by calmodulin (CAM) and identify by mutagenesis the apocalmodulin (apoCaM) and Ca2+-calmodulin (CaCaM) regulatory sites in RyR2. (2) Characterize the regulation of RyR2 by redox active and NO-related molecules and identify regulatory redox-sensitive and S-nitrosylation sites by chemical analysis and mutagenesis. (3) Determine the in vivo role of the above RyR2 regulatory mechanisms by creating mutant ES cells differentiated into cardiomyocytes deficient in RyR2 calmodulin binding and S- nitrosylation sites and mice expressing RyR2s deficient in calmodulin binding and S-nitrosylation sites. The functional properties of normal and mutant RyR2s will be determined in intact cells and with isolated membranes and purified channels under normal and simulated ischemic and postischemic conditions. The functional effects of Ca2+, Mg2+, ATP, pH, calmodulin, and redox-active and NO-related molecules will be assessed in Ca2+ imaging, SR vesicle-Ca2+ flux, [3H]ryanodine binding and single channel measurements. The studies will provide new insights into the complex interaction of the cardiac RyR with its regulatory ligands and how these regulatory processes are altered in the ischemic and post-ischemic heart.